Expression cloning of a human sulfotransferase that directs the synthesis of the HNK-1 glycan on the neural cell adhesion molecule and glycolipids.
The HNK-1 carbohydrate is expressed on various adhesion molecules in the nervous system and is suggested to play a role in cell-cell and cell-substratum interactions. Here we describe the isolation and functional expression of a cDNA encoding a human sulfotransferase that synthesizes the HNK-1 carbohydrate epitope. A mutant Chinese hamster ovary cell line, Lec2, which stably expresses human neural cell adhesion molecule (N-CAM) (Lec2-NCAM), was first established. Lec2-NCAM was co-transfected with a human fetal brain cDNA library, a cDNA encoding the rat glucuronyltransferase that forms a precursor of the HNK-1 carbohydrate, and a vector encoding the polyoma large T antigen. The transfected Lec2-NCAM cells expressing the HNK-1 glycan were enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA encoding a sulfotransferase, HNK-1ST, that directs the expression of the HNK-1 carbohydrate epitope on the cell surface. The deduced amino acid sequence indicates that the enzyme is a type II membrane protein. Sequence analysis revealed that there is a short amino acid sequence in the presumed catalytic domain, which is highly homologous to the corresponding sequence in other Golgi-associated sulfotransferases so far cloned. The amount of HNK-1ST transcript is high in fetal brain compared with fetal lung, kidney, and liver. Expression of HNK-1ST resulted in the formation of the HNK-1 epitope on N-CAM and a soluble chimeric form of HNK-1ST was shown to add a sulfate group to a precursor, GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->R, forming sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->R. The results combined together indicate that the cloned HNK-1ST directs the synthesis of the HNK-1 carbohydrate epitope on both glycoproteins and glycolipids in the nervous tissues.
Ong
E
E
Glycobiology Program, La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037, USA.
Yeh
J C
JC
Ding
Y
Y
Hindsgaul
O
O
Fukuda
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002448:Cell Adhesion / Q000235:genetics; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes / Q000096:biosynthesis; D005333:Fetus / Q000201:enzymology; D015870:Gene Expression; D006017:Glycolipids / Q000096:biosynthesis*; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D019006:Neural Cell Adhesion Molecules / Q000096:biosynthesis*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015238:Sulfotransferases / Q000235:genetics* / Q000378:metabolism; D014018:Tissue Distribution; D014312:Trisaccharides / Q000096:biosynthesis*; D000097811:Carbohydrate Sulfotransferases
D018076:DNA, Complementary; D000939:Epitopes; D006017:Glycolipids; D019006:Neural Cell Adhesion Molecules; D011994:Recombinant Proteins; D015238:Sulfotransferases; D014312:Trisaccharides; C499549:CHST10 protein, human; D000097811:Carbohydrate Sulfotransferases; C499547:Chst10 protein, rat; C109318:HNK-1 sulfotransferase